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Acute lymphoblastic leukemia (ALL) can be classified into different subgroups based on recurrent genetic alterations. Here, targeted RNA-sequencing was used to identify the novel subgroups of ALL in 144 B-other and 40 'classical' ALL samples. The 'classical' TCF3-PBX1, ETV6-RUNX1, KMT2A-rearranged, BCR-ABL1, and novel P2RY8-CRLF2, ABL-, JAK2-, ZNF384-, MEF2D-, and NUTM1-fusions were easily identified by fusion transcript analysis. IGH-CRLF2 and IGH-EPOR were found by abnormally high levels of expression of CRLF2 or EPOR. DUX4-rearranged were identified by the unusual expression of DUX4 genes and an alternative exon of ERG, or by clustering analysis of gene expression. PAX5-driven ALL, including fusions, intragenic amplifications and mutations were identified by SNV analysis and manual inspection using the IGV software. Exon junction analysis allowed detection of some intragenic ERG and IKZF1 deletions. CRLF2-high associated with initial white blood cell (WBC) ≥50,000/L and GATA3 risk alleles (rs3781093 and rs3824662), while ABL/JAK2/EPOR-fusions associated with high WBC, NCI high risk and IKZF1del. ZNF384-fusions associated with CALLA-negativity and NUTM1-fusions with infants. In conclusion, Targeted RNA-sequencing further classified 96/144 (66.7%) B-other cases. All novel ALL subgroups, except for iAMP21, hyper- and hypodiploid cases were identified. Curiously, we observed higher frequencies of girls within B-'rest' ALLs and boys in PAX5-driven cases.

Vaccinium ashei Reade, popularly known as Rabbiteye blueberry, has several therapeutic properties attributed to the phenolic compounds present in its leaves and fruits. Here, we sought to evaluate the effects of the hydroalcoholic extract from V. ashei leaves (Bluegem cultivar, VAB) in T-cell Acute lymphoblastic leukemia (T-ALL). The VAB extract was toxic to T-ALL cells at the ∼60 µg/ml concentration. T-ALL cell death occurred through apoptosis. VAB extract was found to induce micronuclei formation, p53 pathway activation, and cell cycle arrest. Those mutagenic effects were evidenced through microscopy analysis and molecular p53 pathway activation. A series of phenolic compounds were identified in VAB extract by mass spectrometry, such as vanillic acid, catechin, caffeic acid, chlorogenic acid, rutin, coumaric acid, taxifolin, quercetin and naringenin, some of which are presumed to induce DNA damage. In conclusion, the V. ashei leaves extract may have important secondary metabolites with antileukemic properties.

Made available in DSpace on 2021-06-25T10:35:17Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Ministério da Ciência, Tecnologia, Inovações e Comunicações Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs. Departamento de Tecnologia Bioquímico-Farmacêutica Faculdade de Ciências Farmacêuticas Universidade de São Paulo Centro Infantil Boldrini, Campinas Department of Medical Genetics Faculty of Medical Sciences State University of Campinas, Campinas Heart Institute (InCor) Medical School University of São Paulo Biosciences Institute UNESP – São Paulo State University Coastal Campus, São Vicente Department of Parasitology Biomedical Sciences Institute University of São Paulo Department of Clinical and Toxicological Analysis Faculdade de Ciências Farmacêuticas Universidade de São Paulo Biosciences Institute UNESP – São Paulo State University Coastal Campus, São Vicente FAPESP: 2013/08139-8 FAPESP: 2013/08617-7 FAPESP: 2014/06863-3 FAPESP: 2015/07749-2 FAPESP: 2016/25896-5 FAPESP: 2018/15104-0 FAPESP: 2018/15549-1 FAPESP: 2018/18257-1 CNPq: 28/2018 CNPq: 301596/2017-4 CAPES: 309595/2016-9 CNPq: 423532/2018-9

The drug l-asparaginase is a cornerstone in the treatment of acute lymphoblastic leukemia (ALL). The native E. colil-asparaginase used in Brazil until recently has been manufactured by Medac/Kyowa. Then a decision was taken by the Ministry of Health in 2017 to supply the National Health System with a cheaper alternative l-asparaginase manufactured by Beijing SL Pharmaceutical, called Leuginase®. As opposed to Medac, the asparaginase that has been in use in Brazil under the trade name of Aginasa®, it was not possible to find a single entry with the terms Leuginase in the Pubmed repository. The apparent lack of clinical studies and the scarcity of safety information provided to the hospitals by the drug distributor created a debate among Brazilian pediatric oncologists about issues of safety and efficacy that culminated eventually in a court decision to halt the distribution of the new drug all over the country. Boldrini Children's Center, a non-profit pediatric oncohematology hospital, has conducted its own evaluation of Leuginase®. Mass spectrometry analyses found at least 12 different contaminating host-cell proteins (HCP) in Leuginase®. The presence of two HCP (beta-lactamase and malate dehydrogenase) was confirmed by orthogonal methodologies. The relative number of HCP peptides ranged from 19 to 37% of the total peptides identified by mass spectrometry. In vivo studies in mice injected with Leuginase® revealed a 3 times lower plasma bioavailability and the development of higher antibody titres against l-asparaginase in comparison to Aginasa®-injected animals. The decision to buy a new drug based on its price alone is not safe. Developing countries are especially vulnerable to cheaper alternatives that lack solid quality assurance., Highlights • l-asparaginase is one of the first biologicals to be used and an essential drug for the treatment of acute lymphoblastic leukemia (ALL). • Current access to technology has facilitated the worldwide production of l-asparaginase and other biosimilars at lower prices. • Different quality control standards among countries pose a serious challenge to warrant quality, safety and efficacy of biosimilars. l-asparaginase is a cornerstone drug in the treatment of acute lymphoblastic leukemia (ALL). A cheaper l-asparaginase biosimilar was introduced in the Brazilian market in 2017. This report describes the bioanalytical evaluation of such biosimilar, which revealed the presence of at least 12 different contaminating host-cell proteins (HCP), that accounted for 19 to 37% of the peptides identified by three independent mass spectrometry analyses. The second most abundant HCP alone was confirmed by an orthogonal method to represent 2.4% of the total protein mass. Studies carried out in mice showed lower plasma bioavailability and higher anti-asparaginase antibodies titres for the biosimilar in comparison to the well-known Medac l-asparaginase previously used in the Country.

Process related to power generation, i.e., extraction and burning of coal, strongly affects the soil, water resources, and air quality. Pollutants generated by the coal mining can lead to a number of environmental problems and has become matter of global concern. This study has focused on to evaluate the cytotoxicity and phytotoxicity of surface waters collected in the surroundings of the Thermoelectric Power Plant President Medici-UTPM (Candiota, Brazil), through Allium cepa bioassay and physicochemical analyzes. Three water samples collected at points P1, P2, and P3 were compared to the negative control (NC) for all variables analyzed. The P2 sample resulted in the lowest mitotic index (MI) values (23, 33.1, and 40.7%), followed by P1 (23, 33.1, and 40.7%). However, the P3 sample had the highest MI values (50.2, 40.1, and 36.3%) and was the most mutagenic sample compared to NC and other treatments. Samples P1, P2, and P3 were phytotoxic to A. cepa, resulting in abnormal seedlings. Through physicochemical analysis, all collected water samples showed high electrical conductivity and turbidity, indicating an increase in the deposition of metallic ions and organic compounds in these samples. Based on these findings, we can conclude that the analysis of surface water is an important step for screening mining areas potentially contaminated by coal compounds. Therefore, our study points out some consequences of mining process, in terms of A. cepa bioassay as well as their harmful effect on physicochemical constituents of surface waters, indicating the need for environmental monitoring of this region.

Background The spontaneous immortalization of primary malignant cells is frequently assigned to their genetic instability during in vitro culturing. In this study, the new epithelial ovarian cancer cell line CAISMOV24 was described and compared with its original low-grade serous ovarian carcinoma. Methods The in vitro culture was established with cells isolated from ascites of a 60-year-old female patient with recurrent ovarian cancer. The CAISMOV24 line was assessed for cell growth, production of soluble biomarkers, expression of surface molecules and screened for typical mutations found in serous ovarian carcinoma. Additionally, comparative genomic hybridization was employed to compare genomic alterations between the CAISMOV24 cell line and its primary malignant cells. Results CAISMOV24 has been in continuous culture for more than 30 months and more than 100 in vitro passages. The cell surface molecules EpCAM, PVR and CD73 are overexpressed on CAISMOV24 cells compared to the primary malignant cells. CAISMOV24 continues to produce CA125 and HE4 in vitro. Although the cell line had developed alongside the accumulation of genomic alterations (28 CNV in primary cells and 37 CNV in CAISMOV24), most of them were related to CNVs already present in primary malignant cells. CAISMOV24 cell line harbored KRAS mutation with wild type TP53, therefore it is characterized as low-grade serous carcinoma. Conclusion Our results corroborate with the idea that genomic alterations, depicted by CNVs, can be used for subtyping epithelial ovarian carcinomas. Additionally, CAISMOV24 cell line was characterized as a low-grade serous ovarian carcinoma, which still resembles its primary malignant cells. Electronic supplementary material The online version of this article (10.1186/s12885-017-3716-4) contains supplementary material, which is available to authorized users.

Orientador: Katlin Brauer Massirer Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia Resumo: Proteínas de ligação a RNA (RNA-binding proteins - RBPs) regulam pós-transcricionalmente um vasto número de genes. Sabe-se que mutações em RBPs ou alterações na sua regulação estão associadas a doenças neurodegenerativas. Essas RBPs induzem a formação de inclusões patológicas, que frequentemente estão co-localizadas com marcadores de grânulos de estresse (SGs). A proteína Caprin-1 (ou RNG-105) é uma RBP citoplasmática expressa em tecidos de mamíferos e é particularmente abundante nos neurônios. Liga-se a mRNAs-alvos em complexos ribonucleoproteicos mensageiros (mRNPs) regulando o seu processamento. Observou-se que a expressão ectópica (EE) de Caprin-1 induz a formação de SGs. Deste modo, o presente estudo buscou avaliar o mecanismo molecular pelo qual Caprin-1 atua na indução de SGs e compreender a sua função, com enfooque no grupo de RNAs-alvos regulados. Para isso, utilizamos células humanas superexpressando Caprin-1 seguida de análises em larga escala, incluindo: RIP-seq (RNA-immunoprecipitation and sequencing) e RNA-seq (RNA sequencing). Os resultados de RIP-seq mostrou um enriquecimento de 63% de rRNAs e 23% de genes codificadores de proteínas no RIP de Caprin-1 em relação ao controle. Pela classificação funcional do subgrupo de RNAs que codificam proteínas, observamos que muitos constituintes ribossomais e componentes da maquinaria de iniciação da tradução foram enriquecidos. Nós confirmamos a co-imunoprecipitação do 18S rRNA e NPM1, transcritos relacionados com o complexo de iniciação da tradução e biogênese de ribossomos. Digno de nota, em nosso trabalho observamos que Caprin-1 pode estar auto-regulando sua expressão, mas os mecanismos envolvidos ainda não estão claros. Através da co-imunoprecipitação proteica, validamos as interações de Caprin-1 com a RPS3 (proteína da subunidade ribossomal 40S) e com a RBP hnRNPC1 /C2 que atua no processamento de pre-mRNAs, transporte e na estabilização de mRNAs. Pelas análises de transcriptômica, observamos que a EE de Caprin-1 atua de forma global na expressão diferencial de genes. Porém, na classificação funcional dos genes diferencialmente expressos, observamos uma repressão de classes funcionais importantes, tais como constituintes ribossomais, da maquinaria de tradução e do metabolismo celular. Em conjunto, nossos resultados mostram uma correlação entre o papel de Caprin-1 em Grânulos de estresse e no processamento de RNAs. Nesta perspectiva, acreditamos que as interações de Caprin-1 com os RNAs-alvos podem modular os grânulos e a regulação da tradução. Desta forma, o nosso resultado contribuiu na investigação do papel de Caprin-1 em Grânulos de estresse, uma vez que a importância fisiológica da maioria dos alvos de mRNAs identificados ainda não foram esclarecidas Abstract: Caprin-1 (also known as RNG-105) is a cytoplasmic RNA-binding protein (RBP) expressed in mammalian tissues and is particularly abundant in neurons as part of RNA granules. This RBP binds mRNA targets in mRNPs and contains a conserved RG-rich domain, which is related to cellular granules and to reversible aggregation of proteins. Ectopic expression (EE) of Caprin-1 induces assembly of high-density cytoplasmic granules, called stress granules. Because activity and aggregation of RBPs are associated with many neurodegenerative and protein-misfolding diseases, we wanted to know the molecular mechanism by which Caprin-1 mediates stress granule induction and understand its function, focusing on the group of its RNA-targets. Here, we use human HEK293T cells to perform large-scale analysis including: RNA-immunoprecipitation and sequencing (RIP-seq) and RNA-sequencing (RNA-seq) upon Caprin-1 EE. RIP-seq results show 63% of rRNAs and 23% of protein-coding genes enriched in Caprin-1 RIP-seq in relation to control. Among the subgroup of RNA coding genes, most of the target candidates represent constituent of ribosome and components of translational initiation machinery. We have confirmed a handful of Caprin-1 transcripts-target involved in ribosome and translation initiation complex as 18S rRNA and NPM1. Interestingly, here we show that Caprin-1 protein auto-regulates its expression, but the molecular mechanisms have still to be elucidated. Through co-IP, we validated interactions of Caprin-1 with RPS3 and hnRNPC1/C2, ribosomal 40S subunit protein and a RBP that act to stabilize transcripts, respectively. By EE experiments, Caprin-1 is neither a universal repressor nor a universal activator, but some classes involved in translation machinery have been overepresented as repressed in the GO analysis. The combination of our results add to a link between Caprin-1 in stress granules and RNA processing. In this view, we propose that Caprin-1-RNA interactions might modulate stress granule and translation regulation and we expect to contribute to the understanding of its broader involved in neurodegenerative diseases. The next step is investigating the cellular and mechanistic understanding of the Caprin-1-dependent stress granules, since the physiological significance of most of the identified RNA targets has not yet been investigated Mestrado Genética Animal e Evolução Mestra em Genética e Biologia Molecular CAPES FAPESP 2014/20174-6